Method for inducing bone formation using an extract of human osteosarcoma cell line SAOS-2

ABSTRACT

The invention relates to a method for inducing bone formation via administration of an extract of devitalized, freeze dried Saos-2 cells. The extract is prepared by contacting the freeze dried Saos-2 cells, which are devitalized in the act of freeze drying, with a weak denaturing agent of the type used to separate proteins from cells. The extract does have properties superior to comparable treatment using whole Saos-2 cells. The extract is then further treated by applying it to a removing separating gel, and then fractions from the gel which have a molecular weight of from about 10 kd to about 60 kd. Also described are the extract itself, and formulations containing it.

RELATED APPLICATION

This application is a continuation-in-part of application Ser. No.683,283, filed Apr. 10, 1991, which is a continuation-in-part of U.S.patent application Ser. No. 107,299, filed Oct. 9, 1987, now U.S. Pat.No. 5,035,901.

FIELD OF THE INVENTION

This invention relates to a method for inducing bone formation in amammal. More specifically, it relates to such a method in which a boneforming effective amount of an extract of human osteosarcoma cell lineSaos-2 is administered to the subject. Also disclosed is the extractused in the method, and a process for making it.

BACKGROUND AND PRIOR ART

The ability to induce bone formation and growth is an area of scientificinquiry which has seen a great deal of activity over the past 20-25years. Initial observations by Urist et al, Science 150: 893-899 (1965),J. Dent. Res. 50: 1392-1406 (1971), that a substance residing indecalcified bone and now referred to as "bone morphogenic protein"("BMP" hereafter) could induce ectopic cartilage and bone formation viaendochonal ossification after implantation adjacent to skeletal musclesand away from preexisting bone have led to research to isolate and tocharacterize this material, as well as to identify sources for it.Efforts have also been directed to identifying and isolating othermaterials having the same or similar properties. For example, a materialherein referred to as "BIA" for bone inducing agent has been shown toexist in some strains of transformed, cultured human epithelial andosteosarcoma cells. Exemplary of this are Anderson et al. Am. J. Path.44:507-519 (1964) and J. Cell Biol. 33: 165-177 (FL transformed amnioncells); Anderson, et al, Fed; Proc; 27: 475 (1868) (HeLa cells);Wlodarski Exp; Cell Res; 57: 446-448 (1969) (WISH amnion cells);Wlodarski, et al Calcif. Tiss. Res. 7: 345-352 (1971); (neoplasticcells) Amitani et al., Gann 66: 327-329 (1975); Hanamura, et al., Clin.Orthop. & Rel. Res. 148: 274-280 (1980) (Dunn mouse osteosarcoma cellsof BFO strain).

The patent literature in this field as it relates to BMP is growing.U.S. Pat. Nos. 4,294,753; 4,455,256; 4,526,909; 4,563,489; 4,596,574;4,619,989; and 4,761,471, all issued to Urist, arise out of the work hehas done on BMP. The most recent of these patents describes theisolation of BMP from frozen bone samples isolated from cadavers.Essentially, the bone was frozen, defatted, demineralized and freezedried, after which a series of dissolving and dialyzing steps werecarried out, ending in fractionation, dialysis, and SDS-PAGE analysis.The BMP is described as an acidic protein, with molecular weight of17.5±0.5 kd for the human variety, whereas bovine BMP has molecularweight of 18.5±0.5 kd. The patent discusses how it is difficult toseparate the bovine BMP from associated proteins, and how the associatedproteins for both human and bovine BMP are necessary to optimize theeffect of the protein on bone growth.

U.S. Patents have issued to others, as exemplified by U.S. Pat. Nos.4,394,370; 4,472,840; 4,563,350; 4,642,120; and 4,968,590. For the mostpart, these patents deal with grafts for repairing osseous injuries. The'590 patent describes "pure mammalian osteogenic protein" and its uses.The protein, when glycosylated, has a molecular weight of 30 kd onSDS-PAGE, and is obtained from demineralized bone matrix. The bones areobtained from cattle. This protein may be identical to osteogenin, asdescribed by Luyten et al, J. Biochem. 264: 13377-13380 (1989).

As the field has grown, it is convenient to refer to the class ofproteins which have the specified effect as "BMPs". Thus, Wozney et al,Science 242: 1528-1534 (1989), have isolated genes for 3 BMPs, havingmolecular weights of 30, 18, and 16 kds. Recombinant BMPs have beenproduced via transfecting these genes into cultured monkey and hamstercells. Wang et al, Proc. Natl. Acad. Sci USA 87: 2220-2224 (1990),describe bone induction in animal when BMP-2A is implanted in GuHClinactivated, decalcified bone matrix carriers. Sampath et al, J. Biol.Chem. 265: 13198-13205 (1990), describe "osteoinductive protein" (OP),also isolated from demineralized bovine bone. The OP is a 30 kd dimer,having 16+18 kd subunits. This protein also induces bone when implantedwith a GuHCl inactivated bone matrix carrier. Bentz et al, J. Biol. Chem264: 20805-20810 (1989) have described "osteoinductive factor" (OIF)from bovine bone, and characterize it as a 22-28 kd protein which mustreact with TGF-beta to have osteoinductive effect in vivo. Bessho et al,Biochem. Biophys. Res. Comm. 165: 595-601 (1989), also isolated anosteoinductive protein of 18 kd.

Isolating protein from bone is an onerous task, requiring enormousamounts of raw materials for extremely small yields. The amount ofprocessing involved cuts the available yield as well. Further, thesource of the raw material (bone), must generally be animal rather thanhuman. Given the diversity of individuals within an animal species,non-uniformity of protein from batch to batch is to be expected albeitbeing undesirable.

Given the drawbacks discussed supra, the use of cultured cells as asource of bone inducing agents and/or bone morphogenic proteins presentsan attractive alternative. Cell masses can be grown up quickly, and whena cell line is used, one expects the protein to be uniform, since celllines by definition are uniform.

The aforementioned epithelial cell lines, as indicated, areosteoinductive, but they are osteoinductive only when live cells areinjected. The experiments showing this demonstrated efficacy usingimmunosuppressed animals. See Anderson, Connect. Tiss. Res. 24: 3-12(1990); Anderson, et al., Am. J. Patho 44: 507-519 (1964); Wlodarski, etal., Calcif. Tiss. Res. 5: 70-79 (1970); Anderson et al, Fed. Proc. 27:475 (1968). Implantation of live, foreign cells into a subject with ahealthy or non-suppressed immune system will almost inevitably result ina strong, perhaps long term immunological response, so administration oflive cells is not advisable. Also, when the cell line is tumorigenic, asmost cell lines are, the ramifications of administering live cellsinclude serious risks of transplanting neoplasia into the subject.Monitoring the site of implantation with removal at a "critical point"is not possible, due to the risk of malignant invasion by the implant.The immediate suggestion, i.e., to introduce dead epithelial cellskilled by freezing repeatedly into the subject, has proven to beineffective. Devitalized, freeze dried, FL, WISH, and HeLa cells, aswell as their extracts, have not been effective as osteoinducing agents.Anderson et al, Clin. Orthop. Rel. Res. 119: 211-224 (1976).

It is rarely the case that one can obtain an osteoinductive cell linewhich has an effect when the cell line is devitalized. Workers out ofthe lab of Amitani and Takaoka, as per Amitani et al, Gann 66: 327-329(1975); Takaoka, et al., Clin Orthop. Rel. Res. 164: 265-270 (1982);Takaoka, et al., Clin Orthop. Rel. Res. 144: 258-264 (1989), havereported positive results with an osteoinductive BFO strain of Dunnmurine osteosarcoma, and human cell line H-OS-6. The aforementionedlaboratory has not made these cell lines available to other researchers,so this work cannot be verified or compared to results with other celllines. Also, as the art well knows, the odds against securing a secondcell line with properties identical to a first one are astronomical. Tobe denied access to a cell line, even if one is aware of its lineage,renders it impossible for the artisan to repeat reported work. Even inthe case where a researcher reports a detailed, careful protocol for howa cell line was produced, isolated or derived, repetition of this workhardly guarantees reproduction of the initial results. Hence, theexistence of cell banks, depositories, and the general cordiality ofresearchers in sharing cell lines.

When treating humans, it is desirable to treat with material as close tonative human material as possible. U.S. Pat. No. 5,035,901, to Andersonat al., describes that crude extracts of cell line Saos-2 induce boneformation, even in devitalized form. The results reported therein weresurprising because (i) the prior publications in the field suggestedthat dead human cell lines were not osteoinductive, and (ii) comparisonwith many other cell lines showed that the properties of Saos-2 were notshared, either among other human cell lines or among other mammaliancell lines.

The work on Saos-2 has continued. In U.S. patent application Ser. No.683,283, filed Apr. 10, 1991 extracts of Saos-2 not only possessosteoinductive effect but show activity superior to that possessed bythe whole cells. This is surprising because the BIA from Saos-2 cellline has not yet been isolated or characterized. Extraction protocolsare known to damage some molecules, and in the process of extraction,one inevitably loses a portion of the starting material. With this lackof certainty in mind, it was thus more than slightly surprising that anSaos- 2 extract, prepared as described infra, was effective in inducingbone formation in subject animals (i.e., rats) that were immunocompetentand have rejected less purified preparations of BIA.

Further purification has now been carried out, and it has been foundthat extracts of Saos-2 contain, at the least, "bone morphogenic protein4", or "BMP4" as it is referred to hereafter, as well as transforminggrowth factor beta (TGF-β). Surprisingly, these materials are present inthe non-bone-inducing human osteosarcoma cell line designated U2-OS, butin U2-OS cells are insufficient to provoke the bone inducing effectachieved with Saos-2, indicating that the Saos-2 cell line harbors atleast a third, unidentified factor which, in combination with the listedmaterials, leads to the surprising properties characteristic of Saos-2extracts. These new extracts are the subject of the invention and aredescribed in the following disclosure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS EXAMPLE 1

Cell line Saos-2, described by Fogh et al, in Fogh, ed. Human TumorCells In Vitro (Plenum, N.Y. 115-159) (1975) was received as a gift;however, it may also be obtained from public sources, such as theAmerican Type Culture Collection Under Accession Number ATCC HTB 85.This is a non-restricted culture.

The Saos-2 cells were seeded at a density of approximately 3×10⁴cells/cm² in either glass prescription bottles or in 150 cm² plastic "T"flasks. They were grown to confluence in approximately 1 week inDulbecco's MEM, with 10% fetal calf serum, 2.2 mM L-glutamine added,penicillin (20 U/ml), amphotericin (50 ng/ml), and streptomycin (20mg/ml) in 5% CO₂ atmosphere at 37° C. After reaching confluence, thecells were trypsinized, subdivided 1-4 or 1-5 and reseeded for furthercell production. Approximately 2.0×10⁷ confluent cells were releasedfrom one glass flask at time of trypsinization or scraping, andviability, studied by trypan blue exclusion, was 95% or greater.

EXAMPLE 2

Confluent cultures of Saos-2 cells were gently rinsed twice with asterile 0.9% sodium chloride solution to remove medium and stored at-70° C. until needed. For construction of the pellets of devitalizedcells or BIA extracts, the cells were thawed, scraped with a rubberpoliceman and 8.0×10⁷ cell aliquots were pelleted in 50 ml conical tubesby centrifugation at 2000 rpm for 10 minutes. The supernatant wasdiscarded and the cells were defatted by resuspending the pellets in twowashes of acetone. (Each wash was 5 times the volume of pellet). Afteracetone treatment, the cellular material was sedimented at 2000 rpm for10 minutes and air dried at 4° C. for one hour. The resultant pelletswere then freeze-dried, weighed and stored at -20° C. in a sterile, airtight container.

In order to facilitate retention of the pellets at the implantation sitea collagen gel product (Vitrogen, Collagen Corporation, California) wasmixed with the devitalized and defatted Saos-2 cell material or extractsthereof. First, SAOS-2 cells (80×10⁶ cells per implant) were dispersedin 300 μl of 0.01N HCl (40 μl acid per 10×10⁶ cells) for 30 minutes atroom temperature. Then, 700 μl of Vitrogen collagen gel, equal to thevolume of the cell pellet and acid was added to the acidified pelletsuspension. After further mixing, storage at room temperature for 3hours and addition of 300 μl of 0.1M pH7 sodium phosphate buffer (thesame volume as acid) the Saos-2 collagen pellets were stored at -20° C.In some experiments where pellets had been prepared with fewer than80×10⁶ cells, the quantity of collagen added was increased in order tomaintain a relatively constant total volume of 1.4 to 1.6 ml per pellet.The Saos-2 collagen pellets were frozen, lyophilized and stored at -20°C. until implantation.

The freeze-dried cell pellets were used either "as is", or followingextraction protocols. Examples 3 and 4, which follow, describe oneextraction protocol using 4M guanidium hydrochloride (GuHCl), althoughsimilar results, not reported here, were also obtained using 6M urea,all other parameters being the same.

EXAMPLE 3

The defatted, freeze-dried Saos-2 cells were resuspended in 4M guanidiumhydrochloride (Sigma), 0.1 g protein/10 ml of GuHCl (w/v) andcontinuously stirred at 4° C. for 48 hours. After extraction, theresidual particulates were sedimented at 3000 RPM for 10 min. anddiscarded. The supernatant was placed in dialysis tubing with a 6000 to8000 MW cut-off and dialyzed exhaustively against 0.05 M, pH 7.4phosphate-buffered saline (PBS) at 4° C. for 24 to 48 hours. Duringdialysis, a precipitate formed that was resuspended and freeze-dried asdescribed above. Before GuHCl extraction, each defatted and freeze-driedpellet derived from 100×10⁶ Saos-2 cells weighed approximately 100 mg(dry weight).

EXAMPLE 4

Small pellets (0.7×0.3 cm) of freeze-dried defatted cells or ofreprecipitated GuHCl extract of Saos cells were implanted undermetaphane anaesthesia beneath the latissimus dorsi muscles of Nu/Nu mice(obtained from Charles River Labs) and the skin incision was closed bystainless steel staples. At first, animals were sacrificed and implantswere recovered after three weeks, but it was found with experience thatonly ten days to two weeks is required for full expression of cartilage,bone and bone marrow at the implantation site. Similar implants wereprepared using the rat osteosarcoma cell line UMR-106 or the humanosterosarcoma cell line U20S, where, in each case the cells were alsoprepared as indicated supra.

EXAMPLE 5

At the time of sacrifice, the implants were removed, bisected, and onehalf of each implant was fixed for 24 to 48 hours in 10%phosphate-buffered formalin, embedded in paraffin and sectioned andstained by conventional histologic methods without a decalcificationstep. Although the implants showed extensive calcification after only 10days, it was possible to make sections of the implants with reasonablefacility without decalcification. In some instances the ossifiedimplants were fixed in 2.5% glutaraldehyde in cacodylate buffer,post-fixed in osmium tetroxide, embedded in Spurr low-viscosity resinand sectioned at 1 micron thickness with toluidine blue stain for lightmicroscopy or at 40 nM with lead citrate and uranyl acetate stains forelectron microscopy. Electron microscopy was carried out using a ZeissEM10-A electron microscope operating at 80 KV.

The unfixed halves of selected implants were homogenized in N-butanol,and the resultant butanol extract was analyzed for alkaline phosphatasespecific activity following Hsu et al, Biochem. Biophys. Acta 913:329-334 (1987) as a quantitative measure of ossification.

                  TABLE 1                                                         ______________________________________                                        Type of Implant                                                                              no. with Bone/no. without Bone                                 ______________________________________                                        defatted Saos-2 cells                                                                        71/80                                                            reprecipitated GUHCl 31/32                                                    extract of Saos-2 cells                                                       defatted UMR-106 cells 0/8                                                    defatted U2-OS cells  0/14                                                  ______________________________________                                    

EXAMPLE 6

The unique qualities of Saos-2 as an osteoinductive cell line were shownwhen comparative experiments were carried out, using various otherosteosarcoma cell lines. Specifically, human osteosarcoma cell linesU2-OS, TE85 and MG63 were tested. The first two cell lines are availablefrom the ATCC (HTB96, CRL 1543), while MG63 was a gift.

Following the same protocols for cell culture, preparation of pellets,and implants, the three cell lines listed above, plus UMR-106 ratosteosarcoma cells, and Saos-2 were tested in a side by side comparison.Six mice were tested using UMR-106 (8 pellets), 2 mice for MG63 (14pellets), 3 mice for U2-OS (3 pellets), and thirteen mice (17 pellets)for Saos-2. Following cervical dislocation and analysis of the pelletswhen removed, all Saos-2 pellets showed bone, and some were very hard.The UMR-106 U2OS MG-63 and TE-85 pellets remained soft, showing no bonecartilage or marrow formation.

The failure of any osteosarcoma cell line other than Saos-2 to elicitbone formation led to the decision not to proceed with furtherextraction experiments on these cell lines.

EXAMPLE 7

An additional experiment was carried out to determine whether defatting,i.e., the use of acetone, was required for efficacy.

Either extraction with acetone or freeze drying will devitalize a cell,and in these experiments, cells were simply devitalized via freezedrying. Freeze drying, it will be recalled, only removes moisture fromthe cell sample.

Again, following the general protocols described surra, cells were grownup, pelleted, and implanted, the only difference being that in one setof experiments, no acetone extraction took place. These are thenon-defatted cell lines.

In the following Table 2, the results for formation of bone, cartilageand marrow formation are presented summarizing the results ofexperiments on 3 animals. In this Table "+++" indicates much induction,"++" moderate induction, "+" a small amount, and "0" none whatsoever.

                  TABLE 2                                                         ______________________________________                                        Animal No. Bone        Cartilage                                                                              Marrow                                        ______________________________________                                        DEFATTED                                                                           1         +++         ++     ++                                            2 ++ ++ +                                                                     3 +++ ++ +                                                                  NON-DEFATTED                                                                       1         ++          +      +                                             2 + + 0                                                                       3 + + +                                                                     ______________________________________                                    

EXAMPLE 8

Further purification of the Saos-2 derived material was carried out.Specifically, 100-500 mg samples of Saos-2 pellets, prepared asdescribed supra, were dissolved in 25 volumes of 4M GuHCl, again asdescribed supra. These materials were then centrifuged at 25,000 rpm, toseparate cellular debris therefrom. Following removal of the debris, theresulting supernatant was applied to a Pharmacia Sephacryl-200 column(5×100 cm), pre-equilibriated with 4M GuHCl. Fractions in the molecularweight range of 10-60 kd were removed, pooled, and dialyzed againstphosphate buffer, using 10,000 kd cutoff dialysis tubing. The dialyzedretentates were then pooled, lyophilized, and prepared for implant. Inthese experiments, 500 mg samples of dried Saos-2 pellets yielded about25-35 mg of proteinaceous material.

EXAMPLE 9

The materials described in Example 8 were then used in in vivoexperiments. Twenty adult male Long Evans rats were divided into 4groups of 5 rats each. Each rat was treated to create a bone injury.Specifically, a lateral incision was performed to expose the rightfemoral diaphysis, and a 5-hole, 1.5 mm AO plate was then placed on theanterior surface, using four cortical screws. The plates were then swungaway from femurs, and a 4 mm central diaphysial defect was created,using a water cooled burr.

The four groups were then divided into a control (group 1), a grouptreated with pure bovine collagen (group 2), a group treated with anautograft obtained by morselizing resected femur segments (group 3), anda group treated with 10 mg of the extract of Example 8, combined with 10mg of pure bovine collagen (Example 4). The implants for groups 2, 3 and4 were packed into one half of a #4 gelatin capsule, slid over thedistal cut end of femur. The plate was secured, and hemostatic closureperformed. Animals were radiographed at four and eight weeks. After fourweeks, two animals were sacrificed from each group at random, and thethree remaining animals were sacrificed after eight weeks. Two sets ofexperiments were carried out. Coronal sections through the defect weredecalcified, and stained with hematoxalin and epsin.

The results are summarized in Table 3, which follows. Control animalsshowed non-union at four or eight weeks, as the defects were filled withfibrous tissue and/or skeletal muscle, and there was only a trace of newbone at resecton edges, and subperiosteally. The second group (collagenonly), also showed no healing at either four or eight weeks. Even ateight weeks, unresorbed residual collagen and fibrous tissue spanned thegap, with only a small quantity of new bone near cut ends. Group 3animals (autograft) showed notable callous formation, but there wasnon-union in all four and eight week animals, with most of the graftedparticles of bone being gradually resorbed over eight weeks.

Group 4, utilizing the combination of collagen and extract, showed earlyunion, with cartilage containing calluses at four weeks, and completeunion by lamellar bone, with a reformed medullary canal by eight weeks.None of the groups exhibited inflammation.

                  TABLE 3                                                         ______________________________________                                        PROMOTION OF BONE REPAIR                                                                  FIBROUS            PERCENT                                          NON- BONY WITH                                                                UNION UNION BONY UNION                                                      ______________________________________                                        No implant  5          0       0                                                Collagen implant 5 0 0                                                        Autograft 5 0 0                                                               BIA plus collagen 5 4 80%                                                   ______________________________________                                    

EXAMPLE 10

Given the osteoinductive properties of Saos-2 cell line extract, it wasof interest to determine if the cell line expressed any known bonemorphogenetic proteins. The analysis was based upon a study of mRNA inthe cells. Specifically, total RNA was prepared from exponentiallygrowing Saos-2 cells, by lysing samples in 0.5% SDS in 10 mM EDTA (ph8.0), followed by acidification with 0.1 M Na acetate (pH 2.2). Thelysate was then treated with H₂ O, equilibriated with ethanol to extracttotal RNA. The extracted RNA (20 ug) was size fractionated using agarosegel electrophoresis, blotted onto nylon membranes, and then hybridizedwith ³² P labelled cDNA probes for known osteoinducers "BMP2", "BMP3",and "BMP4", using standard Northern blotting. Similar experiments werecarried out using cDNA for TGF-β. The results revealed that both BMP-4and TGF-β mRNA were present in the Saos-2 cells, but no RNA for BMP-2and BMP-3 was found.

EXAMPLE 11

An experiment similar to that carried out on Saos-2 was performed oncell line U2-OS. The expression pattern was identical, i.e., both BMP4and TGF-β mRNA were found in U2-OS, but neither of BMP-2 or BMP-3 werefound. In both of Examples 10 and 11, the tests with anti-TGF-β antibodyshowed a stronger reaction with two high molecular weight proteins (65kd, 72 kd), than with any material of the known molecular weight ofTGF-β, i.e., about 12.5 kd. This suggests that some intermediate form ofTGF-β is present, although this is by no means certain.

The foregoing examples also demonstrate that bone induction was obtainedwhen extracts of devitalized Saos-2 cell line were applied to the siteof injury. Results were obtained not only with extracts obtained viatreatment with "weak denaturing agents", i.e., those materials known topermit proteins to refold from whatever conformation is possessed by thedenatured protein to the active form, but also from extracts which havebeen further treated, i.e., subjected to purification on an absorbing orseparating gel. Specifically, those fractions in the molecular weightrange of from about 10 kd to about 60 kd are effective. "Weak denaturingagents", as the term is used herein, is identical to that used Builder,e.g., U.S. Pat. No. 4,511,502, the disclosure of which is incorporatedby reference. In addition to the GuHCl and urea solutions describedherein, other solvents possessing the recited properties will be knownto the skilled artisan, and need not be repeated here.

The absorbing or separating gel, as used herein, means any gel to whicha protein containing molecule will adhere to when a solution passingthrough the gel contains the protein. "Sephacryl" was used in theexperiments, however, any of the standard gels used in proteinseparation such as Sepharose, etc., may be used in its stead.

In experiments not reported herein, molecules of a larger molecularweight were also tested, but failed to induce ectopic bone formation inanimals, using the same model reported herein. Thus, the smallermolecular weight fraction of the Saos-2 extract is clearly the activeportion.

The analyses of osteoinductive factor mRNA in the cell extracts issignificant. The results show that both of Saos-2 and U2-OS expressBMP-4 and TGF-β; however, it is also shown, supra, that U2-OS does notinduce ectopic bone formation. Thus, as of yet at least one unidentifiedfactor is believed to contribute to the osteoinductive properties ofSaos-2. This factor does not appear to be present in the otherosteosarcoma cell lines tested, as these do not show osteoinductiveproperties.

Thus, the invention is a method for inducing bone formation in a mammal,such as a human, by administering or treating the subject mammal in needof bone formation or healing with an amount of an extract ofdevitalized, freeze dried Saos-2 cells sufficient to promote bonehealing or bone formation, wherein said extract is prepared bycontacting freeze dried Saos-2 cells with a solvent containing a weakdenaturing agent to form a supernatant and a sediment, separating thesupernatant therefrom and treating the supernatant by contacting it to aseparating gel, and removing 10-60 kd fractions from the gel. The 10-60kd fractions contain the bone inducing activity and may be furthertreated by, e.g., dialyzing against a salt solution with a low molecularweight cut-off point. A "low molecular weight cut-off" is e.g.,6000-8000 kd. It will be understood that the specific dialysis stepdescribed herein, and other methods for concentrating the activematerial, available to the skilled artisan, including other dialysisprotocols are optional. Following dialysis, the 10-60 kd "retentate" maybe further treated to form a precipitate, pellet, solution, lyophilisateand so forth. This precipitate may be pelleted and freeze dried, andadministered to the subject mammal in that form.

Also described herein is the precipitate used in the treatment of thesubject immunocompetent animal, prepared as described surra as are thefreeze dried, devitalized Saos-2 cells per se. The extract, i.e., theprecipitate, can be further treated to form a pellet, or can be combinedwith a pharmaceutically acceptable carrier to form a composition usefulin inducing bone formation in a mammal.

As with the devitalized Saos-2 cells, the extract described herein canbe used in the treatment of mammalian and veterinary diseases andneoplasms. An example of the latter is the use of the extract in therepair of large fractures or displacements, such as in equine andrelated species. Such fractures admit to treatment via splinting and/orbone replacement, using the extract described herein, either alone or incombination with biodegradable materials or pharmaceutical carriers.Similar efficacy is envisioned for, e.g., repair of deviated limbs incanine and other related species, as well as spine fusion.

The extract may be useful for systemic as well as local treatment toaugment bone growth, to prevent bone loss in metabolic diseases such asosteoporosis, and to speed fracture healing and bone repair and bonereplacement via systemic administration. In short, pathologicalconditions which result in general or specific bone loss, such asosteoporosis can be treated using the materials described herein. Otherconditions which may be usefully treated include the replacement of boneremoved for tumor surgery, for maxillofacial repair, to speed rigidfusion of vertebral bodies in spinal surgery ("slipped discs,"scoliosis, etc), bone grafting, to speed healing of traumatic fractures,to augment bonding of resected bone surfaces to porous, biocompatibleprostheses, to effect repair of non-uniform fractures, and so forth. Anyof the conditions which the art speaks of being treatable by BMP or BMPsmay be treated with the subject extract.

The materials described herein are preferably combined with collagen toform material suitable for implant or other in situ administration. Thecollagen may be bovine collagen, or collagen derived from some otherspecies, preferably one identical to the subject being treated, or froman evolutionarily related species. Autologous collagen is alsopreferred, as is collagen that has been treated in some way to render itnon-immunogenic.

It will be understood that the specification and examples areillustrative but not limitative of the present invention and that otherembodiments within the spirit and scope of the invention will suggestthemselves to those skilled in the art.

What is claimed is:
 1. Method for inducing bone formation in a mammalcomprising administering an amount of an extract of Saos-2 cellssufficient to promote bone healing or bone formation to a mammal in needof said bone healing or bone formation, said extract of Saos-2 cellsobtained by contacting a sample of devitalized, freeze dried Saos-2cells with a denaturing agent in an amount and concentration to permitrefolding of any denatured protein to active form or insufficient todenature protein to form a supernatant and a precipitate, applying saidsupernatant to a protein absorbing gel, and removing protein fractionsfrom said absorbing gel having a molecular weight range of from about 10kd to about 60 kd, to form an extract of Saos-2 cells.
 2. Method ofclaim 1, wherein said denaturing agent is a solution of guanidiumhydrochloride at a concentration which permits a protein to refold toactive form.
 3. Method of claim 1, wherein said denaturing agent is asolution of urea at a concentration insufficient to denature a protein.4. Method of claim 2, wherein said guanidium hydrochloride is at aconcentration of about 4M.
 5. Method of claim 3, wherein said urea is ata concentration of about 6M.
 6. Method of claim 1, wherein saiddevitalized Saos-2 cells are defatted.
 7. Method of claim 1, whereinsaid mammal is in need of healing of a bone defect treatment of a bonetumor or of promotion of assimilation of a bone graft.
 8. Method ofclaim 1, further comprising lyophilizing said extract prior toadministering to said mammal.
 9. Method of claim 1, comprising combiningsaid extract with collagen prior to administering to said mammal. 10.Method for inducing bone formation in a mammal comprising administeringan amount of an extract of Saos-2 cells sufficient to promote bonehealing or bone formation to a mammal in need of said bone healing orbone formation, said extract of Saos-2 cells obtained by contacting asample of devitalized, freeze dried Saos-2 cells with a denaturing agentin an amount and concentration to permit refolding of any denaturedprotein to active form or insufficient to denature protein to form asupernatant and a precipitate, removing said supernatant from saidprecipitate, resuspending said precipitate in a solution, treating saidsolution to form a second supernatant and a second precipitate, whereinsaid second precipitate is said extract.
 11. Extract of Saos-2 cellsuseful in inducing bone formation, said extract prepared by contacting asample of devitalized freeze dried Saos-2 cells with a solution of adenaturing agent in an amount and concentration to permit refolding ofany denatured protein to active form or insufficient to denature proteinto form a supernatant and a precipitate, separating said supernatanttherefrom, applying said supernatant to a protein absorbing gel andremoving fractions from said protein absorbing gel having a molecularweight of from about 10 kd to about 60 kd.
 12. Extract of claim 11,wherein said denaturing agent is a solution of guanidium hydrochloride.13. Extract of claim 11, wherein said denaturing agent is a solution ofurea.
 14. Extract of claim 12, wherein said solution of guanidiumhydrochloride is at a concentration of about 4M.
 15. Extract of claim13, wherein said solution of urea is at a concentration of about 6M. 16.Extract of claim 11, in solution form.
 17. Extract of claim 11, inpellet form.
 18. Extract of claim 11, in lyophilized form. 19.Composition useful in inducing bone formation comprising the extract ofclaim 11 and a pharmaceutically acceptable carrier.
 20. Composition ofclaim 19, wherein said pharmaceutical carrier is biodegradable. 21.Composition of claim 20, wherein said biodegradable pharmaceuticalcarrier is a matrix.
 22. Composition useful in inducing bone formationcomprising the extract of claim 11 and collagen.